Characterization and expression analysis of SnRK2, PYL, and ABF/ AREB/ ABI5 gene families in sweet potato

Abscisic acid (ABA) signaling in plants is essential to several aspects of plant development, such as tolerance to environmental stresses and growth. ABA signaling is also important for storage organ formation in crops, such as sweet potato. However, the repertoire of I. batatas ABA signaling gene families has not yet been fully characterized, so that it is unclear which members of these families are necessary for tuberization. Therefore, genome-wide identification of the sweet potato ABF/ AREB/ ABI5, SnRK2, and PYL gene families was performed, along with phylogenetic, motif, cis-regulatory element (CRE), and expression analyses. Nine ABF, eight SnRK2, and eleven PYL gene family members were identified, and there was high sequence conservation among these proteins that were revealed by phylogenetic and motif analyses. The promoter sequences of these genes had multiple CREs that were involved in hormone responses and stress responses. In silico and qRT-PCR expression analyses revealed that these genes were expressed in various tissues and that IbABF3, IbABF4, IbDPBF3, IbDPBF4, IbPYL4, IbSnRK2.1, and IbSnRK2.2 were significantly expressed during storage root development. These results are an important reference that can be used for functional validation studies to better understand how ABA signaling elicits storage root formation at the molecular level.


Introduction
Abscisic acid (ABA) is a major plant phytohormone that is involved in diverse processes which include growth, development, and adaptation [1].For instance, ABA is involved in seed development and germination, the adaptive response to environmental stressors (such as temperature extremes, salinity and drought, UVB radiation, and pathogens), flowering, vegetative growth, and seed dormancy [2,3].
In order for ABA to elicit its effects, various ABA-dependent signalling pathways are activated.Major components of these pathways include: the PYRABACTIN RESISTANCE (PYR/ PYL/RCAR) ABA receptors, PROTEIN PHOSPHATASE 2C (PP2C), Sucrose Nonfermentin-g1-Related Protein Kinase 2 (SnRK2), NADPH oxidases, ion channels and downstream transcription factors (TFs) [3].One model of ABA-dependent signal transduction suggests that ABA is required to bind to the PYR/PYL/RCAR receptor so that PP2C can bind to the PYR/PYLs and thus inactivate PP2C [3].This inactivation causes SNF1-type kinases to become active and target TFs to regulate gene expression and ABA-dependent ion channels [3].TFs bind to cis-elements in the promoter regions of ABA-responsive genes [3].Some of these ciselements include ABA-responsive elements (ABREs), the G-box, CE3, motif III, and hex3 [3].
The (ABA)-responsive element binding proteins/ABRE-binding factors (AREB/ABFs) are a subfamily of transcription factors (TFs) that are involved in the ABA-dependent signalling pathways.AREB/ABFs are the most important TFs involved in the ABA-dependent ABRE signalling pathways [4].The AREB/ ABF/ ABI5 family of TFs are a subfamily of the bZIP superfamily of proteins.There are two motifs in the bZIP domain: a basic domain that acts as a DNA binding site, and a leucine zipper involved in TF dimerization [4].These TFs have three conserved regions at the N-terminal, designated as C1, C2, and C3 [4].
Sweet potato is an economically important crop that is cultivated for its edible root tubers.The mechanism of tuberization in this crop is not fully understood.An enhanced understanding of this process is important to breed better-yielding varieties.ABA has been previously reported to be involved in the development of sweet potato tubers [12].There is a peak in ABA concentration in the sweet potato root tuber at tuber initiation [13].Additionally, ABA is involved in various processes during sweet potato tuber development, such as promoting starch deposition [14] and regulating tuber thickening by activating cell divisions in meristems [15].This raises the question as to whether there are specific ABA signalling genes that are crucial for sweet potato tuber initiation.
In sweet potato, homologs of the AREB/ABF family were found to be up-regulated during drought conditions [16] but their roles in tuberization have not been thoroughly investigated.Dutt et al. [17] noted that StABF2 and StABF4 are potential genes that can be overexpressed by genetic engineering to improve potato tuber yield.Two ABF genes (AtABF2 and AtABF4) were shown to positively regulate tuber induction in transgenic potato plants, but only StABF4 was found to improve tuber yield without stunting growth in other plant parts [18].Additionally, StABF1 expression was found to increase under tuberizing conditions in potato [19].In potato (Solanum tuberosum), AtABF4-expressing plants had increased tuber quality and quantity [20].Furthermore, StABI5-like 1 (StABL1) promotes early potato tuber maturity via interactions with FLOWERING LOCUS T (FT) homologs and regulation of GA 2-oxidase 1 genes [21].It is therefore important to investigate whether the sweet potato homologs of these genes have similar functions during storage root formation.
This paper is the first report of the characterization of the PYL and SnRK2 gene families in sweet potato, and the expression analyses of these families and the AREB/ ABF/ ABI5 subfamily during storage root formation.The results of this study serve as a basis for the further study of ABA signalling in sweet potato and can be used for further study to improve breeding programs.The most promising gene candidates from this study can be functionally validated in the future to better understand the roles of ABA signalling during storage root initiation and how this impacts yield.

Plant material
Whole sweet potato root tubers (cultivar O49) were obtained from the germplasm collection at The University of the West Indies Field Station (St.Augustine, Trinidad and Tobago).This cultivar is a popular one that is grown in Trinidad and Tobago [22] and the characteristics of this cultivar during storage root initiation and bulking have been previously described [23].These root tubers were planted singly in 25 cm diameter pots in sandy soil and left for seven weeks.The plants were watered during the day with 500 mL water as required.The plants were fertilized with NPK (12:24:12) fertilizer as directed by the manufacturer.Samples of the storage roots, pencil roots, fibrous roots, leaves, and green stems were harvested at 49 days after transplantation (DAT).Storage root samples at 35 DAT were also collected.Three biological replicates were collected for each tissue.All the samples were used immediately after harvesting.

Identification of ABA signalling genes in I. batatas genome
The bioinformatics analyses were conducted on the Galaxy server (https://usegalaxy.eu/)unless otherwise specified [24].All genes were named according to their homology to the A. thaliana genes, followed by Chromosome location.
AREB/ABF/ABI5 gene family.The nine AREB/ ABF/ ABI5 A. thaliana protein sequences were obtained from the UniProtKB database [25] (https://www.uniprot.org/)and were used as query sequences against the sweet potato proteome [26] (www.sweetpotao.com) in a BLASTP search [27] with a threshold e-value of 1e -10 .The putative protein sequences were examined in the NCBI CDD [28] for the presence of a complete bZIP domain (cd14707: bZIP_plant_B-ZIP46).The protein sequences were also examined in InterPro [29] (https://www.ebi.ac.uk/ interpro/search/sequence-search) for the bZIP domain (IPR043452) and the presence of a DNA binding site and the dimer interface that is characteristic of these proteins (cd14707).The 11 resulting sequences were searched against the nr BLASTP database and any hits that corresponded to G-box binding factor (GBF) proteins were excluded.
PYL and SnRK2 gene families.The 14 A. thaliana PYL and 10 SnRK2 protein sequences were used as queries in a BLASTP search against the sweet potato proteome with e-value of 0.001 and the top 20 hits for each sequence were displayed.The hits were checked for complete domains in the CDD and any hits with the wrong domain were discarded.The SnRK2 hits were also checked in PfamScan (https://www.ebi.ac.uk/Tools/pfa/pfamscan/) for the presence of the protein kinase domain (PF00069).PfamScan was used to check that the PYL proteins had the typical PYL domain (PF10604).Any hits with incomplete domains were used as TBLASTN queries in the I. batatas NCBI Transcriptome Shotgun Assembly (TSA) database to find complete open reading frames for the protein.

Analysis of cis-acting regulatory elements in promoter sequences
The 2 kb region upstream of the gene sequences were extracted using bedtools [36] and were analysed in PlantCARE [37] for the presence of cis-acting regulatory elements (CREs) (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/).Only hits from the sense strand were accepted.

In silico RNA-Seq expression analysis of ABA signalling genes
Raw RNA-seq reads were downloaded from the NCBI Sequence Read Archive (SRA) (https:// www.ncbi.nlm.nih.gov/Traces/study/) and the National Genomics Data Center (NGDC) (https://ngdc.cncb.ac.cn/).The raw data (PRJCA000640) from Ding et al. [38] was downloaded to investigate the expression of the ABA signalling genes in various tissues.The raw data (PRJNA491292) from Wu et al. [39] was used to compare the expression of the ABA signalling genes in storage roots vs. fibrous roots at 30, 40, and 50 days after transplantation (DAT).The time-course datasets (PRJNA647694) of sweet potato roots at different stages of development (fibrous roots [stages S4 and S8], pencil roots [stages S10 and S12], and initiating tuberous roots [stages S14 and S16]) were also used [40].To investigate the effects of ABA treatment, salicylic acid (SA) treatment, and methyl jasmonate (MeJA) treatment relative to a control, the PRJNA511028 dataset was downloaded from the NCBI SRA.The raw reads were aligned to the genome with STAR [41] and the read counts were obtained from featureCounts [42].Differential expression was analysed with DESeq2 [43].The FPKM values were plotted on a heatmap using TBTools [44].

RNA isolation and qRT-PCR expression analysis
Total RNA was isolated from the plant tissue using the second protocol described by Gromadka et al. [45] with the modification that standard acidified phenol-chloroform RNA extraction was used.The RNA pellets were dissolved in 100% formamide and stored at -20˚C.RNA quantity and quality were checked on a Nanodrop 2000 spectrophotometer (Thermo Scientific) and via agarose gel electrophoresis.First strand cDNA synthesis was performed with the SuperScript IV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's directions.Primers were designed using IDT PrimerQuest (S1 Table ) (https://www.idtdna.com/pages/tools/primerquest)and synthesized by Macrogen Inc. (Seoul, Korea).The genes that were investigated were chosen based on their significant differential expression in the in silico datasets.
The cDNA was diluted twofold and 1 μL of cDNA from three pooled biological replicates was used in each qRT-PCR reaction.The 50 μL qRT-PCR reaction mixture had final concentrations of 1X Power SYBR 1 Green Master Mix (Invitrogen, Carlsbad, CA, USA), 200 nM forward primer, and 200 nM reverse primer.Each reaction was run in triplicate on a qTower3 thermal cycler (Analytic Jena).The expression levels were normalized to that of the COX housekeeping gene [46] and analysed using the 2 -ΔCT method.Statistical analyses (One-way ANOVA followed by Duncan's multiple range post-hoc test (p-value � 0.05)) were conducted with IBM SPSS Statistics version 29.

Prediction of ABA signalling PPI network, Gene Ontology analysis, and KEGG enrichment
The sweet potato proteome in the study was annotated for protein-protein interactions (PPIs) in the STRING-db [47] (https://string-db.org/).The predicted PPI network for the ABA signalling genes were extracted from this network.The ABA signalling proteins were submitted to ShinyGO v. 0.77 (http://bioinformatics.sdstate.edu/go/)and KOBAS-i (http://kobas.cbi.pku.edu.cn/) for Gene Ontology (GO) and KEGG analyses, respectively.The Ipomoea triloba and Ipomoea nil database annotations were used for each analysis, respectively.

Identification of I. batatas ABA signalling genes
The I. batatas ABA signalling genes that were identified and characterized in this study are shown in Table 1.The genes were named based on their sequence similarity with the A. thaliana sequences and by ascending order along the chromosome.Nine IbABF/ AREB/ ABI5 sequences were identified on six chromosomes with 2-9 exons, pIs ranging from 4.58-9.49,protein MWs ranging from 21.41-50.34kDa, and they were all predicted to be found in the nucleus.
Twelve PYL gene sequences were discovered in BLASTP.Of these 12 sequences, 2 sequences (g9406 and g25480) showed an incomplete domain.TBLASTN of these 2 sequences against the sweet potato transcriptome shotgun assemblies in NCBI was used to yield the corrected versions of these proteins with the full PYL domain (see S1 File for protein sequences used).One sequence (g55782) only yielded hits that were similar to g55788 so this sequence was discarded, giving a final total of 11 IbPYL genes (Table 1).These 11 sequences had 1-4 exons (Fig 1 ), and the proteins encoded by these genes ranged from 185-244 a.a. in length, and were predicted to localize to the nucleus, chloroplast, and cytoplasm.
The eight IbSnRK2 genes had coding sequences comprised of 8-10 exons, and these sequences encoded proteins that were 306-371 a.a. in length, had a narrow pI range from 4.67-6.29,and were all predicted to localize in the nucleus.

Motif analysis of I. batatas ABA signalling proteins
The results of the MEME analysis to find conserved motifs in the I. batatas ABA signalling proteins are depicted in Ten conserved motifs were found in the IbPYL proteins and there were similar arrangements of these motifs in the sequences (Fig 2B).Motifs 1, 2, and 4 were present in all the IbPYL proteins and these motifs, along with Motif 3, represent the START-like domain (IPR023393).
There were also ten conserved motifs with similar arrangements in the IbSnRK2 proteins (Fig 2C).Motifs 1, 2, 8, and 9 were present in all 8 IbSnRK2 proteins.Motifs 1-5, and Motif 7 represent the protein kinase domain (IPR011009) that is characteristic of these proteins.

Phylogenetic analysis of I. batatas ABA signalling proteins
The IbABF/ AREB/ ABI5 protein sequences clustered into three phylogenetic subfamilies (Fig 3A ), with 4, 3, and 2 IbABF/ AREB/ ABI5 sequences in Subfamilies I, II, and III respectively.Subfamily I members had the unique Motif 9 in their sequences.
The IbPYL protein sequences were grouped into three subfamilies in a phylogenetic tree (  Subfamilies II and III had 8-10 exons.There were no motifs that were specific to any subfamily (Fig 2C ).

Analysis of CREs in the promoter sequences of ABA signalling genes
The CREs that were identified in the ABA signalling gene sequences are shown in S2 Table .All the ABA signalling genes had multiple light-responsive elements.There were also several CREs present that are involved in hormone responsiveness.The most commonly occurring hormone-responsive CRE was the MeJA-responsive CRE, which was present in 21 of the 28 sequences.Fifteen of these sequences had at least one ABA-responsive CRE (ABRE).Less than half of the sequences had CREs for responsiveness to auxin, GA, SA, and ethylene.There were also several CREs present that are involved in stress responses, development, and protein binding.Some of the common developmental CREs were as-1 (involved in root-specific expression), HD-Zip 1 (involved in palisade mesophyll cell differentiation), and CAT-box (involved in meristem expression).There were numerous CREs involved in stress and defence responses such as ARE (anaerobic induction), DRE core (drought responsiveness), LTR (low temperature responsiveness), MBS (drought inducible), STRE (stress responsiveness), and wound responsiveness (W box, WUN-motif, WRE).There are similarities between the expression of some of the genes in Figs 5 and 6.For example, IbABF2, IbPYL2, IbPYL6, and IbSnRK2.5 are significantly down-regulated and IbPYL4 is significantly up-regulated in both datasets.There were also some differences, since IbABF4 and IbSnRK2.2 are up-regulated in Fig 6, but the former is not differentially expressed, and the latter is down-regulated in Fig 5.
The in silico expression of the ABA signalling genes in response to the application of ABA, MeJA, and SA in FRs, leaves, and stems of sweet potato cultivar Xushu18 are shown in Fig 7.For each hormone treatment, there was some tissue-dependent variations in the expression patterns.The largest number of DEGs were observed in response to ABA (Fig 7A).IbABF2 and IbABF4 were significantly up-regulated in response to ABA treatment in all three plant parts, whereas IbPYL6 was down-regulated in response to ABA treatment in all three plant parts.IbABF3 and IbSnRK2.5 were significantly up-regulated in stems in response to ABA while IbPYL1 and IbPYL5 were significantly down-regulated in stems after ABA treatment.In leaves, IbABF1 was significantly up-regulated and IbPYL5 was significantly down-regulated in response to ABA treatment.In FRs, IbPYL1 and IbPYL4 were significantly down-regulated in response to ABA treatment.Compared to the previous two hormone treatments, there were more down-regulated ABA signalling genes in response to SA (Fig 7C).Only IbABF2 (in the stem) and IbABF4 (in the leaf and stem) were up-regulated, while IbPYL5 and IbPYL6 (in the stem) and IbPYL4, IbSnRK2.5, and IbSnRK2.8 (in the FR) were down-regulated in response to SA.The ABF/AREB/ABI5 subfamily showed the most up-regulated genes in response to ABA, MeJA, and SA, when compared to that of the PYL and SnRK2 families, which showed down-regulation in some cases.

qRT-PCR expression analyses of ABF/AREB/ABI5, PYL, and SnRK2 genes
The results of the qRT-PCR expression analyses are illustrated in Fig 8 .The expression of the six ABA signalling genes that were investigated were highest in the leaf than in other tissues.were similar in SRs vs. FRs in the qRT-PCR results while there were significant differences in Fig 6 .The expression of IbABF4 at 35 DAT in SRs was significantly higher than that in the other plant parts, excepting the leaf), and this was similar to the higher expression of IbABF4 in SRs vs. FRs that was seen in Fig 6.

Predicted PPI network of ABA signalling genes
The predicted ABA signalling PPI network that was obtained from STRING is illustrated in Fig 9 .A densely connected network was obtained with several of the ABA signalling genes showing high confidence predicted interactions (interaction score > 0.7) with each other, as opposed to individual clusters of genes.While all the IbABF/ AREB/ ABI5 and IbSnRK2 proteins had predicted interactions with each other, only three of the 11 IbPYLs had predicted interactions in the network.These IbPYL proteins (IbPYL-1, -9, and -11) showed high confidence predicted interactions with IbSnRK2.3 and IbSnRK2.6 and medium confidence predicted interactions with IbSnRK2.2,IbSnRK2.8,IbABF5, and IbABI5.
GO analysis revealed that these proteins were annotated to GO terms involved in plant responses to various environmental stimuli (S1 Fig) .These GO terms include: 'regulation of stomatal closure', 'response to carbon dioxide', 'pollen maturation', 'cellular response to light intensity', 'response to water deprivation', 'response to salt stress', and 'response to acid chemical'.The gene list was also enriched in the salt/ drought/ osmotic stress pathway of the 'MAPK Signaling Pathway-Plant' KEGG pathway and the ABA signalling pathway in the 'Plant Hormone Signal Transduction' KEGG pathway.These annotations lend further support for the predicted roles of these proteins in the plant's response to environmental stresses.

Discussion
ABA signalling is vital for several aspects of plant development, including abiotic stress responses, dormancy and germination, regulation of stomatal movements, and vegetative growth [48,49].ABA is also involved in the formation and development of stem tubers and root tubers in various crops [50].Although there has been progress in understanding ABA signalling during tuberization in potato [3], this process is less well-defined in I. batatas.Therefore, this study characterized the repertoire of the ABF/ AREB/ ABI5, PYL, and SnRK2 gene families to gain a better understanding of how they are expressed during storage organ formation.

I. batatas ABA signalling proteins are highly conserved
The intron-exon arrangements of the coding sequences were similar to that of ABA signalling genes from other crops such as potato [3], soybean [11], rice [10], and cotton [8].The high degree of conservation of these sequences were also reflected in their domain organization and phylogenetic relationships.The nine IbABF/ AREB/ ABI5 protein sequences clustered together with their A. thaliana bZIP protein homologs, as also described by Liu et al. [51].Liu et al. reported 11 IbbZIP Group A members, while we reported 9 members since, as described earlier, we excluded any AtGBF homologs from the results, since GBF proteins are not involved in the ABA signalling pathway.Dro ¨ge-Laser et al. [52] identified 13 Group A AtbZIPs, but four of these (AtbZIP-13, -14, -27, and -40) were either uncharacterized, a GBF4 protein, or flowering locus D (and its paralog).Since these four bZIPs are not part of the ABA signalling pathway, they were not used as search queries.
The 11 IbPYL protein sequences were clustered into three subfamilies (Fig 3B ) and this phylogenetic arrangement was also observed in other studies [8][9][10].The clustering of SnRK2 proteins in grape [53] and sugarcane [54] into three subfamilies is consistent with this study.The IbSnRK2 proteins had a highly conserved N-terminal protein kinase domain, and the Cterminus was more variable, which is consistent with previous reports that the variability of the C-terminus contributes to the functional diversity of these proteins [54].
The SnRK2 proteins contained most or all of the 10 motifs that were identified in this family (Fig 2 ), indicating that this family was highly conserved.However, none of the members of the PYL family or the ABF/AREB/ABI5 subfamily contained all 10 identified motifs, with few of the members having identical motif arrangements.This dissimilarity in the motifs present amongst these gene family members may be associated with divergence of the sequences that may lead to functional variation.
The similarity of expression of some of the genes from the same phylogenetic subfamily (e.g.IbDPBF3 and IbDPBF4) indicate that there may be some functional redundancy among homologous gene pairs.However, the expression differences between the majority of genes within a subfamily indicate that some of these genes have unique functions, which contribute to the diversity of ABA-mediated responses in the plant.

ABA signalling during I. batatas storage organ formation
ABA is an important regulator of storage organ development, but there is a lack of studies that investigate the specific roles of ABA during tuberization [50].In sweet potato, ABA is involved in cambial activity in the meristem and regulation of starch deposition [12,14].Chen et al. [50] summarized the current state of knowledge of the ABA signalling genes during storage organ formation.They found that there was both up-and down-regulation of ABFs, PYLs, and SnRK2s during storage organ formation in lotus (Nelumbo nucifera), carrot, radish, and Panax notoginseng [50].There was a lack of corresponding data for crops that form storage roots, such as cassava and sweet potato [50].Therefore, this study sought to fill this gap in the current understanding of storage root formation.
The up-regulation of several ABA signalling genes during SR formation suggests that these genes are likely to have various roles during the tuberization process.There were some similarities and differences in the expression of these genes in sweet potato and that of their homologs in S. tuberosum.For example, StABF1/AREB2 is up-regulated as tuberization progresses [19], but its homolog, IbABF2, is down-regulated during SR development (Figs 5 and 6).The significant expression of IbABF4 during SR formation (Figs 6 and 8) is supported by the finding that StABF4 overexpression led to increased yield in transgenic potato plants through regulation of ABA-GA crosstalk [18,20].Chen et al. [50] discussed how ABA and GA have antagonistic effects that mediate tuberization in several crops.Liu et al. [3] found several genes from the StABF/ AREB/ ABI5 gene family were up-regulated in potato stolons at the onset of tuberization and similar results are observed for I. batatas, where IbABF3, IbDPBF2, IbDPBF3, and IbDPBF4 are up-regulated during SR initiation (Fig 5).StABL1 also regulates potato tuberization by interacting with the StSP6A tuberigen and altering GA metabolism [21].The up-regulation of the StABL1 homologs, IbDPBF3 and IbDPBF4, warrants further studies to determine whether these genes promote SR initiation via a mechanism similar to that in potato [21].There is a paucity of previous studies that determine the functions of PYL and SnRK2 proteins during storage organ formation.Previous research found that the expression levels of Auxin Response Factor 4 (ARF4) and ARF10 genes involved in SR initiation are positively correlated with MePYL1 and MeSnRK2.6 in cassava (Manihot esculenta) and negatively correlated with MePYL2 [55].Therefore, the significant up-regulation of IbPYL4, IbSnRK2.1, and IbSnRK2.2 warrant further investigation.

ABA signalling is involved in sweet potato development
The sweet potato ABA signalling genes have other roles during plant development.The expression of these ABA signalling genes throughout the plant in various plant parts (Fig 4) highlight their importance in various aspects of plant development.An interesting finding was that there was high expression of some of the ABA signalling genes in leaves (Fig 8) although this was not observed for most genes in the in silico dataset (Fig 4).This may be due to a cultivarspecific difference or due to the difference in planting conditions.These ABA signalling genes are important in the sweet potato response to various hormones ( Fig 7).Our observation of the stronger responsiveness of ABF/AREB/ABI5 genes in response to ABA (when compared to that with other hormones) was corroborated by a previous study [51].
Most of the IbSnRK2 genes did not show differential expression in response to hormone treatments, and this was observed in potato, where StSnRK2 genes did not show much obvious responses to ABA [56].The StSnRK2 genes showed significant changes in response to salt and osmotic stress, so that the IbSnRK2 genes are likely to be more involved in responses to stress than in responses to hormones.It is interesting to note that IbSnRK2.1 and IbSnRK2.2, which was up-regulated during SR development (Figs 5 and 6 respectively), did not show differential expression in response to ABA.These two genes are part of Subfamily I (Fig 3) which contain the ABA-independent SnRK2 proteins.Subfamilies II and III contain the ABA-dependent SnRK2 proteins [57].Conversely, IbSnRK2.5 is significantly down-regulated during SR formation (Figs 5 and 6) and is a member of the ABA-dependent Subfamily II.IbSnRK2.5 may therefore be an important negative regulator of sweet potato SR formation in response to ABA.
Except for IbPYL2, all the differentially expressed IbPYLs were down-regulated in response to ABA, MeJA, and/or SA (Fig 7).A similar expression pattern was observed in potato, where several StPYLs had lower expression after ABA treatment [58].Interestingly, most of these StPYLs showed up-regulated expression in response to auxin [58].
The high degree of connectivity in the predicted PPI network suggests that these proteins regulate multiple developmental pathways in a highly coordinated manner.Several IbPYL, IbPP2C, and IbSnRK2 genes were differentially expressed in transgenic sweet potato plants that had stronger low-temperature stress tolerance than wild-type plants [59].Solis et al. [60] found an IbAREB gene was up-regulated during drought stress.Additionally, Wang et al. [61] found that IbABF4 confers resistance to several abiotic stresses in sweet potato and this gene was used to investigate drought tolerance in several Caribbean varieties of sweet potato [62].IbPYL8 forms part of a complex that mediates ABA-dependent drought responses [63].The presence of CREs involved in stress and defence responses in the promoter sequences of the I. batatas ABA signalling genes (S2 Table ) suggests that the proteins that are encoded by these genes are vital to the plant's responses to various biotic and abiotic stresses.
This study lays a comprehensive framework that can be used for future studies on the functional validation of these genes.The well-studied AtSnRK2.-2,-3, and -6 are considered core components of the A. thaliana ABA signalling network [64] so future experiments are required to determine if this is also true of the I. batatas homologs, IbSnRK2.3 and IbSnRK2.6.The predicted STRING PPI network indicated that IbSnRK2.3 and IbSnRK2.6 are the only members of this gene family that have high confidence interactions (confidence score > 0.7) with both PYL and ABF proteins (Fig 9 ), as opposed to the other SnRK2 proteins which either showed medium or low confidence interactions with PYL proteins.Additionally, the Subfamily II AtSnRK2 proteins (AtSnRK2.7 and AtSnRK2.8)regulate drought-responsive genes, such as AtABFs, to enable drought tolerance [65].The high sequence conservation in the SnRK2 family suggests that there may be some conservation of function as well.AtAREB1, AtAREB2, and AtABF3 are Subfamily I members that are thought to be master regulators for ABA-dependent expression under water stress [66].The corresponding I. batatas homologs (IbABF-1, -2, -3, and -4) are likely to also be involved in similar functions, as already experimentally shown for IbABF4 [61].Several MeABF, MePYL, and MeSnRK2 genes were up-regulated in cassava during multiple abiotic stresses and the sweet potato homologs likely have similar roles [67,68].
The correlations between the in silico and qRT-PCR datasets give further support for the importance of these genes during storage organ formation.A limitation of using datasets from different cultivars is that there are more factors involved in the variations in gene expression that were observed so that a specific reason for the differences in gene expression could not be identified.

Conclusions
In this study, 9 IbABF/ AREB/ ABI5, 11 IbPYL, and 8 IbSnRK2 genes were identified and characterized from the I. batatas genome.The physicochemical properties, domain organizations, and phylogenetic analyses of these sequences revealed that these sequences are highly conserved.There were numerous cis-regulatory elements involved in light responsiveness, hormone responsiveness, developmental responses, and defence and stress responses in the promoter sequences of these genes.In silico expression analyses revealed that several of these genes were differentially expressed during SR initiation and development.Similar expression patterns were observed with qRT-PCR.The predicted PPI network for these proteins indicated that these ABA signalling proteins form a highly connected network.The up-regulation of IbABF3, IbABF4, IbDPBF3, IbDPBF4, IbPYL4, IbSnRK2.1, and IbSnRK2.2 during SR development makes them ideal candidates for further investigations on their roles during SR initiation.

Fig 2 .
There were 10 conserved motifs found in the ABF/ AREB/ ABI5 proteins (Fig 2A)and the motif order was conserved among the sequences.Motif 2 was the only motif present in all nine IbABF sequences.All nine sequences had a combination of one or more of Motifs 1, 3, and 4. Motif 1 represents the IPR043452 bZIP domain that is characteristic of these TFs.
Fig 3B) with 2, 3, and 6 IbPYLs belonging to Subfamilies I, II, and III respectively.IbPYLs with similar domain organization clustered in the same subfamily.For example, IbPYL1 and IbPYL11 from Subfamily 1 have the unique Motif 8 (Fig 2B), while IbPYL-2, -3, -7, -8, -9, and -10 belonged to Subfamily III and contained the unique Motif 5. Subfamilies I and II have IbPYLs that were encoded by genes with one or two exons only (Fig 1) while Subfamily III IbPYLs were encoded by genes with three or four exons only.The IbSNRK2 proteins were grouped into three phylogenetic subfamilies I, II, and III (Fig 3C), with 3, 3, and 2 IbSnRK2s respectively.Subfamily I contained IbSnRK2 proteins that were encoded by coding sequences with nine exons, while the coding sequences for members of

Fig 2 .
Fig 2. Motifs in the ABA signalling proteins discovered using MEME.The motif arrangements are shown for the (a) ABF/ AREB/ ABI5 family, (b) PYL family, and (c) SnRK2 family.A maximum of 10 motifs were searched with motif length between 10-50 residues.Motif occurrence was set to zero or one occurrence per sequence.https://doi.org/10.1371/journal.pone.0288481.g002

Fig 5 .Fig 6 .
Fig 5. Heatmap illustrating the expression of ABA signalling genes in I. batatas roots at various stages of development.The heatmap illustrates the DEGs at different time points during sweet potato SR development obtained using RNA-seq data from He et al. [40].The stages that were compared were fibrous roots (S4, S8), pencil roots (S10, S12), and initiating storage roots (S14, S16).Pink boxes represent up-regulation and green boxes represent downregulation.There was no significant fold change for the white boxes.Only differentially expressed ABA signalling genes are shown.The criteria for differential expression are |log 2 FC| � 1 and adjusted p-value � 0.05.https://doi.org/10.1371/journal.pone.0288481.g005

Fig 7 .
Fig 7. Expression heatmaps showing the fold changes of ABA signalling genes in sweet potato cultivar Xushu18 in response to different hormone treatments.(a) ABA treatment (b) MeJA treatment (c) SA treatment.Differentially expressed genes (DEGs) were identified using the criteria of log 2 FC � 2 and p-value < 0.05.DEGs are denoted by an asterisk and genes whose expression was too low to calculate a fold change are shown as grey boxes.Red boxes represent up-regulation while blue boxes represent down-regulation.https://doi.org/10.1371/journal.pone.0288481.g007

Fig 8 .
Fig 8. qRT-PCR results of the expression of ABA signalling genes in different I. batatas tissues.Tissue samples were collected at 49 DAT (unless otherwise stated for the SR sample at 35 DAT).The relative expression was calculated by normalization of the expression values to the COX housekeeping gene.Error bars represent the standard deviation of three replicates.Bars not sharing a letter show statistically significant differences (p-value � 0.05) using Duncan's multiple range test.(GS-green stem; PR-pencil root; SR-storage root; FR-fibrous root; L-leaf).https://doi.org/10.1371/journal.pone.0288481.g008

Fig 9 .
Fig 9. Predicted PPI network for I. batatas ABA signalling proteins.The nodes are coloured by protein family (blue-ABF/ AREB/ ABI5; green-PYL; red-SnRK2).The edge thickness is directly proportional to the confidence score of the predicted interaction.Only predicted interactions with a minimum medium confidence score of 0.4 are displayed.The disconnected nodes are hidden from the network.https://doi.org/10.1371/journal.pone.0288481.g009